Methods of measuring potential for therapeutic potency and defining dosages for autologous cell therapies

ABSTRACT

Autologous bone marrow cells (BMC) are transplanted to a heterologous site in a patient after a sample of the patient&#39;s BMC has been tested and found to have a phenotypic profile which meets minimum criteria for transplantation. The phenotypic profile may be obtained by screening a sample of bone marrow cells (BMC) from the patient for the phenotypic profile, such as a CD profile, the phenotype profile may be assessed to determine the likelihood that the BMC will be suitable for transplantation to the heterologous tissue site without enriching particular phenotypic population(s) of the BMC.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 14/526,222,filed on Oct. 28, 2014, which claimed the benefit of ProvisionalApplication No. 61/896,450, filed Oct. 28, 2013, the full disclosures ofwhich are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Introduction to Pharmacogenomics and Personalized Medicine.Pharmacogenomics is the field of selecting the right drug for the rightpatient at the right time. Its genesis was the belief that thepharmaceutical industry would identify and match patients with specificgenetic phenotypes and gene expression profiles who would respond tospecific therapeutic medicines, providing a new era of personalizedmedicine. This would inherently enhance the drugs therapeutic profile,resulting in more successful clinical trial results as the selectedpatients would have already been identified as potential responders tothe therapy. An example of a successful drug that has been developedusing the concept of pharmacogenomics is the drug Herceptin. It iscurrently used in the treatment of early stage Human Epidermal growthfactor Receptor-2 (HER-2) positive and late stage HER-2 positivemetastatic breast cancer where an overexpression of the protein markerHER-2 is caused by a gene mutation in the cancerous cell. This geneticmutation is present in one out of every five breast cancers. Herceptinspecifically targets HER-2 to kill the cancer cells and is often usedwith chemotherapy to decrease the risk of cancer recurrence. Therefore,patient testing HER-2 positive is a pre-specified requirement fortreatment with Herceptin.

The concept of selecting patients who are predisposed to respond toparticular therapies has tremendous potential. A number of efforts havebeen put forth in the development of gene expression assays tocharacterize cancer tumors based on their gene activity—i.e., upregulation or down regulation of specific genetic markers or a geneticmutation specific to the type of cancerous cells. These gene expressionassays would allow a more accurate understanding of the cause of thedisease from a genetic standpoint which would result in the ability topinpoint the specific pathway via which these genes are getting up ordown regulated or mutated. Therefore, a patient's responsiveness toexpensive and often dangerous chemotherapy that is not specificallytailored to the patient's individual needs, could be evaluated inadvance of the therapy which would reduce cost but above all, wouldprevent the patient from going through unnecessary painful treatments.Genomic Health's OncotypeDx gene expression assay is an example of sucha product which is generating millions of dollars per year as adiagnostic assay to determine whether or not a patient has a highlikelihood of responding to therapy. Other new products on the market,CareDx's Allomap assay and CardioDx's Corus test are designed todetermine whether or not a patient should be subjected to a moreinvasive assay to assess the status of the disease. For CareDx's Allomapassay, gene expression analysis of the circulating white cells in theblood is intended to determine if the patient's circulating immunesystem is calm or angry. If calm, the patient does not need to have aninvasive heart biopsy and may even have their dosage of potentiallydangerous immunosuppressant drugs lowered. If angry, the patient needsto have a biopsy, which, if positive, is likely to result in anincreased immunosuppression regimen. For the CardioDx Corus test, theblood based leukocyte gene expression signature is used to safely andquickly help identify whether a patient presenting with chest pain islikely suffering from obstructive coronary artery disease and wouldrequire a coronary angiogram to look for a diseased artery or a heartattack. These are merely three examples of a new field of endeavor inwhich patient specific information is used to help determine appropriatenext steps for informed patient care management. This may and oftentimesdoes include significant and expensive clinical care decisions, andthese tests therefore have value in both mitigating the risks ofunnecessary and potentially dangerous procedures and reducing medicalcosts.

Introduction to Autologous Tissue Therapies. In medicine, there are manyautologous therapies in which a tissue or material is extracted from apatient and is returned to the patient for therapeutic purposes.Autologous therapies can be classified as homologous and non-homologous.Homologous autologous therapies constitute harvesting a tissue from onepart of the body and using it to serve the same basic function whetherit is in the same or a different part of the body of a patient. Examplesof homologous autologous therapies include the harvest of skin from aless visible part of the body to perform a graft on the face of a burnvictim; the use of bone from one part of the body to help reconstructanother more important and critical bone structure; and the extractionof cartilage from one part of the body and its ex vivo culture andexpansion for re-implantation into the body later. Non-homologoustherapies are those in which the harvested tissue and preparationsthereof either by concentration, selection, expansion, modification byadding ligands, culture or any combinations of these are intended toserve a different purpose than the tissues were serving at their time ofharvest. This does not mean they may not mimic natural processes, butrather that they were harvested and are being repurposed from theirstatus at time of harvest. Non-homologous cell therapies include thedelivery of cells or tissues to treat cardiovascular disease, autoimmunediseases, diabetes, metabolic disorders, and a broad variety of cancers.

Cardiovascular indications include acute myocardial infarction, chronicmyocardial ischemia, heart failure of ischemic etiology (with activeischemia or chronically infarcted without the presence of activeischemia) or non-ischemic etiology, or cardiac arrhythmias, refractoryangina, dilated cardiomyopathy. U.S. Pat. No. 7,097,832 issued toKornowski and U.S. Pat. No. 8,496,926 issued to de la Fuente describesuch therapeutic strategies to treat cardiovascular disease using bonemarrow derived cells. Autologous non-homologous tissues that have beeninvestigated preclinically and clinically for such non-homologouscardiac therapeutic purposes are extensive and include bone marrowcells, bone marrow or peripheral blood mononuclear cells, bone marrow orblood derived CD34 cells (whether harvested from the bone marrow or fromthe blood after GCSF stimulated release), bone marrow or blood derivedCD133 cells; bone marrow or blood derived CD 19 cells, bone marrow orblood derived ALDH bright positive cells, bone marrow derived and exvivo expanded mesenchymal cells, bone marrow derived and expandedmesenchymal precursor or progenitor cells, adipose tissue derived cells,and umbilical cord derived cells. Other non-homologous tissues that havebeen used preclinically include c-kit+ cells, placental-derived cells,human amniotic mesenchymal stem cells. Many of these autologoustherapies have been indicated for the treatment of other diseases suchas chronic limb ischemia and intermittent claudication as well asautoimmune and inflammatory diseases such as inflammatory bowel diseasesof ulcerative colitis, Crohn's disease, lupus, osteoarthritis, diabetesand kidney diseases.

The use of activated T-cells, tumor infiltrating leukocytes, andactivated dendridic cells have an expanding role in immunotherapy todayand often involve removing dendritic cells or t cells from the patientand modifying them to enable them to more accurately and aggressivelyattack tumor cells when they are re-administered to the patient.Dendritic cells are antigen-presenting cells, (also known as accessorycells) of the mammalian immune system. Their main function is to processantigen material and present it on the cell surface to the T cells ofthe immune system. Dendritic cells act as messengers between the innateand the adaptive immune systems. T cells or T lymphocytes are a type oflymphocyte (a type of white blood cell) that play a central role incell-mediated immunity. They can be distinguished from otherlymphocytes, such as B cells and natural killer cells (NK cells), by thepresence of a T-cell receptor (TCR.,) on the cell surface. They arecalled T cells because they mature in the thymus (although some alsomature in the tonsils). There are several subsets of T cells, each witha distinct function.

Tissue based autologous therapies, by their nature are personalized anddepend on a number of variables including the patient's age, sex, race,weight, extent of cardiovascular disease progression and the presence ofcomorbidities such as diabetes, hypertension, angina, hyperlipidemiaamong others. All patients do not have the same cells in the sameconcentrations with the same genes or gene expression profiles. Allpatients do not have the same therapeutic potential in their tissues fora given clinical indication nor will all patients respond to a therapyidentically even if the therapeutic potential of a given dosage form isidentical. This is a critical problem in the field.

Different approaches have been used to try and solve various aspects ofthis problem, with particular focus on cardiovascular disease, theleading cause of death in the western world. One potential solution isto attempt to make autologous cell therapy as close to a purepharmaceutical preparation as possible. Examples of such autologous celltherapy products for just cardiovascular applications include Baxter'sor Neostem's selected CD34 cells in chronic myocardial ischemia andacute myocardial infarction, respectively; CellProThera's cultureexpanded CD34+ cells; Miltenyi's selected CD133 cells), culture-expandedMesenchymal Stem Cells (MSCs), Dental Pulp Stem Cells (DPSCs), Cardio3Biosciences culture expanded MSCs with ex vivo stimulateddifferentiation, Capricor's culture expended cardiosphere derived cells,autologous culture expanded CD271 positive cells, and expandedHematopoietic Stem Cells (HSCs), and the like. Such autologous celltherapy products require extensive manipulation and analysis of thecells intended for treatment before they are returned to the patient toenable dosage formulations which are as standardized as possible. Thecell manipulation often requires isolating cells with specific cellsurface markers such as CD34 or CD133 from bone marrow or peripheralblood using antibodies bound to magnetic beads or other standardpurification methods and expanding those isolated cell populations inculture outside of the patient's body to reach the desired dosageformulation required for treatment. That dosage could be determinedbased on patient weight or could be a same standardized dosage in termsof total number of cells to be delivered. Cell analysis usually includesperforming functional assays such as mitogenic or colony forming unitassays (CFU-F, CFU-EC, CFU-HILL, etc.) on these cells to determine thecells' health and their proliferative potential. These types ofextensive manipulation, cell culture handling and analysis significantlyraise the costs of these proposed therapeutic candidates. Not only dothe manipulations themselves have significant labor and complex reagentsand materials costs, but they also have shipping and quality controlcosts on each patient's tissues, resulting in a separate manufacturinglot for each and every dosage form for one particular patient. Eachassay on a potential autologous therapeutic dosage formulation and eachstep in its handling add significantly to the costs of the therapy—whichin turn means that the developer and manufacturer will ultimately haveto charge a higher price for their product. This approach however onlyaddresses one issue by attempting to normalize the dosage in everypatient but still does not result in identical dosages for thesepatients as we can only suggest that the cells performed similarly inthe functional assays involved and passed the lot release testingspecified by the developer for purity,functional assay, sterility andlack of contamination by infectious agents. This does not mean that thecells would behave the same way when re-injected in the patient. Oneexample of an autologous cell therapy product for cardiovascularapplication that employed this selected cell strategy with a successfulclinical trial outcome lies in Baxter's CD34 autologous cell therapy.The double-blind, prospective, randomized, placebo-controlled Phase IItrial was designed to determine the tolerability, efficacy, safety, anddose range of intramyocardial injections of G-CSF mobilized autologousCD34 cells for a reduction of angina episodes in patients withrefractory chronic myocardial ischemia (ACT34-CMI). The results of thistrial published in Losordo D W et al. 2011 [Circ Res. 109:Intramyocardial, Autologous CD34+Cell Therapy for Refractory Angina]have shown that percutaneous intramyocardial injections of mobilizedautologous peripheral blood derived CD34+ cells at dosages of 10⁵cells/kg led to significant improvements in angina frequency andexercise tolerance.

Other purified cells with specific cell surface markers isolated fromthe bone marrow, include the CD19 bone marrow derived cells which havebeen shown to have potential efficacy that is superior to that of wholebone marrow or other cell subtypes in preclinical studies (Goodchild Tet al. 2009, J Am Coll Cardiol Intv. 2: 1005-16). These cells aredescribed in U.S. Pat. No. 7,695,712.

A second approach to the problem is to increase the uniformity oftherapeutic cells delivered to each patient and eliminate the costs ofanalyzing the cells and of manufacturing a separate lot for each dosageformulation through the development of allogeneic tissue dosageformulations so that the tissue from one donor can be applied tothousands of patients. This strategy was previously in development byOsiris Therapeutics with Mesenchymal Stem Cells, and is currently beingdeveloped by Mesoblast with Mesenchymal Precursor Cells. Mesenchymalstem cells (or precursor cells or progenitor cells) have long beenargued to be immune privileged cells such that the allogeneic donorcells will not be attacked by the circulating immune system of therecipient, thus making the possibility of a universal donor a realisticand extremely attractive one. In addition to being obviously more costeffective and commercially viable, this strategy, also has the advantageof enabling the selection of healthy young donors whose healthy cellshave been shown to be much more efficacious that the older diseasedpatients' cells. Other groups including Capricor Therapeutics, Inc., aclinical stage biotechnology company that has migrated to developingallogeneic cardiac stem cells (CSC) after reporting results fromautologous cells, has postulated that their allogenic CSC are not immuneprivileged as the allogeneic mesenchymal stem cells. The Capricorallogenic CSC are rejected or eliminated from the heart, but the cellsare reported to have their therapeutic benefit before being rejected bythe immune system of the recipient or otherwise eliminated. The problemwith this therapeutic approach even if the theoretical assertions on theimmune privilege status of mesenchymal stem cells or time course ofaction preceding rejection for CSCs hold true, is that each lot of cellswill come from a different universal donor or a combination of donorsand they will not be identical. Therefore, to a lesser extent, as in thefirst approach above, one should expect variations in response and inbeneficial outcomes with each donor or batch lot and at present, onecannot presume these variations to be insignificant or predictable.

These current approaches for specific strategies for cell therapies arebased on the classical pharmaceutical mindset of purification tooptimize a dosage form that is the same for all patients, even if it isan autologous therapy. While this has value for advancing the status ofour scientific knowledge and adds significant rigor to cell basedtherapies, it also adds enormously to the complexity of therapies whensimpler approaches described herein are possible. Both of theseapproaches are intended to overcome what is seen as limitations withrespect to the delivery of minimally processed bone marrow (indevelopment by T2Cure, Harvest, Biomet, Thermogenesis, and others) oradipose stromal cells or adipose derived regenerative cells (CytoriTherapeutics and others) which are viewed as more variable from patientto patient.

US Patent Publ. 2010/0127342 describes how gene expression profiles fromautologous cells could be developed to select patients for a giventherapeutic strategy based on their likelihood of being responders tosuch a therapy. This is hereby included by reference, along with thereferences cited therein. Personalized medicine strategies forautologous tissue based therapeutics have significant potential that isnot well appreciated as the diagnostic assessment of a patient'spotential responsiveness can also be an analysis of the autologousdosage form's therapeutic potency.

Signature to Gate Dosage of Autologous Bone Marrow Cells for CardiacRepair in a setting of Heart Failure of Ischemic Etiology, with orwithout Evidence of Active Ischemia. Delivery of autologous mononuclearcells derived from the bone marrow has been shown to be consistentlysafe in the setting of chronic heart failure, chronic myocardialischemia, and acute myocardial infarction. However, while there isconsensus about the safety of these cells, efficacy results from similarclinical studies in the same patient population appear to beinconsistent. Dosage is believed to be a primary culprit driving theseinconsistencies. Consistent with this hypothesis, is the observationthat trials with lower dosages of these cells, whether by design or dueto delivery via the coronary artery route of administration have fewerpositive trial results, and studies with higher cell dosages havegreater rates of success. However, it is noted that higher dosage doesnot necessarily correlate to greater efficacy. This was observed in theBaxter study (Losordo et al. 2011) using mobilized autologous peripheralblood derived CD34+ cells in a patient population with chronicmyocardial ischemia for the treatment of refractory angina whereby thesmallest CD34+ cell dosage of 10⁵ cells/kg had the most significantpositive efficacy results. Variations between patients in cell potencyand in cell dosage delivered may also result in failed therapeuticefficacy trials.

SUMMARY OF THE INVENTION

The embodiments in this invention comprise of (1) the selection ofpatients for autologous tissue therapies based on the analysis of smalltissue samples from the patients in advance of the therapy. Such tissuescan be bone marrow, blood, tumors, muscle, heart tissue, and or othertissues known to be a viable source for autologous therapeutic cells;(2) the development of in vitro diagnostic assays in which a smallamount of tissue can be assessed to see if the source tissue meetsdefined thresholds of cellular, protein and/or genetic characteristicsthat would potentially increase a patient's likelihood of being anenhanced responder to the autologous therapy.

For example, the in vitro diagnostic assay for autologous bone marrowderived mononuclear cells for the treatment of heart failure of ischemicetiology may be specified based on the CD profile of the constituentcells; (3) the development of in vitro diagnostic assays in which asmall amount of tissue can be assessed to see if the source tissue meetsdefined thresholds of cellular, protein and/or genetic characteristicsthat increase a patient's likelihood of achieving a targeted therapeuticdosage when these same cells are harvested, concentrated, andre-administered at a later time point; (4) the delivery of an effectivedosage based on the number of cells from a specific population, proteinsand/or genes that is present in a patient that would qualify the patientas a responder to the autologous therapy; (5) the delivery of aneffective therapeutic dosage based on the efficiency of the deliveryroute and the delivery method inclusive of the delivery system used toinfuse the effective therapeutic dosage into a patient; (6) the deliveryof an effective therapeutic dosage based on the patient's likelihood ofhaving a targeted therapeutic dosage when using autologous point of caretherapies that have significantly reduced costs and also enhanced safetydue to the minimalist nature of the processing. Point of care systemscan concentrate a particular cell population by cell density usinggravity centrifugation. Many other possible means of activating cellsand platelets with short term incubation with other agents, stimulatingthem using energy insertion such as heating or stimulating them withradiation across all the wavelengths possible or energy extraction suchas cooling are possible and may be used in combination with other meansof modifying tissues in a short period of time at the patient's bedsidein a relatively closed system to reduce the potential contamination ofthe therapeutic tissues being modified. The preferred embodiment in thisinvention would preferably be a combination of (5) and (6) but furtherembodiments in this invention can also be any combination(s) of theembodiments discussed herein.

In a first aspect of the present invention, a method for screening apatient to determine a likelihood that the patient's autologous bonemarrow cells (BMC) will be suitable for transplantation to aheterologous tissue site comprises providing a sample of BMC from thepatient. A phenotypic profile of the BMC in the sample is obtained andassessed to determine the likelihood that the BMC will be suitable fortransplantation to the heterologous tissue site without enrichingparticular phenotypic population(s) of the BMC beyond concentrating thecells.

The phenotypic profile is typically a CD profile, and the CD profileusually includes the concentration of cells having at least two CDmarkers in the sample. Often, the CD profile will include theconcentration of cells having at least three CD markers in the sample.The at least two CD markers may be selected from the group consisting CD14, CD19, CD34, CD45, CD133, and CD271 typically including at least CD34and CD133, and often including each of CD19, CD34, and CD133. Inspecific examples, a sample having at least 38500CD34⁺ cells/ml and atleast 6500 CD133⁺ cells/ml is considered suitable for heterologoustransplantation for cardiac conditions in which a delivery efficiency ofat least 18% as measured one hour after delivery can be achieved. Inother specific examples, a sample having at least 13,000 CD19⁺ cells/ml,at least 38,500 CD34⁺ cells/ml, and at least 6,500 CD133⁺ cells/ml isconsidered suitable for heterologous transplantation for cardiacconditions in which a delivery efficiency of at least 18% as measuredone hour after delivery is achieved. The presence of CD and other cellsurface markers may be conveniently assessed by comprises flow cytometryor other convention techniques.

In a second aspect of the present invention, a method for transplantinga patient's bone marrow cells to the patient comprises obtaining bonemarrow cells (BMC) from the patient, where a sample of the patient's BMChas previously been tested and found to have a phenotypic profile whichmeets a minimum criteria for transplantation; and transplanting the BMCso obtained to a heterologous site in the patient. Any of the methodsdiscussed above with respect to screening patient autologous BMC's maybe used to identify BMC's suitable for transplantation.

The autologous BMC are typically transplanted without isolation orenrichment based on phenotype, for example without isolation orenrichment based on the presence of particular CD antigens. Theautologous BMC are particularly suitable for transplanting into cardiactissue, but can also be transplanted into other heterologous tissuesites, in including the limbs for the treatment of critical limbischemia, as well as solid tissues, organs, membranes, blood, and thelike. While the BMC will usually not be enriched or isolated withrespect to their phenotype, they will often be enriched and otherwiseprepared for transplantation in conventional ways without regard tophenotype. When transplanted into cardiac tissue, transplanting oftencomprises intra-myocardial injection, e.g. with a helical needle, andthe cardiac tissue will usually be infarcted where the BMC will be ableto promote regeneration of the infarcted cardiac tissue. Typically, atleast 72,600 CD133⁺ cells are present in the target heart tissue onehour after transplantation and/or at least 420,000CD34⁺ cells arepresent in the infected tissue one hour after transplantation, and/or atleast 144,000 CD19⁺ cells are present in the infarcted tissue one hourafter transplantation. Preferably, at least 72,600 CD133⁺ cells, atleast 162,000 CD34⁺ cells, and at least 144,000 CD19⁺ cells are presentin the target tissue one hour after transplantation. Target tissue willtypically be ischemic such as in a patient with ischemic heart failure,where injection can be into the infarct, the viable tissue, or theperi-infarct region depending on the indication.

Cells are often counted using flow cytometry relative to a standardmarker on these cells such as CD45. Cells identified in theCD45/CD34/CD133 panel, CD45+/CD34+ or CD45+/CD133+ are hematopoieticstem/progenitor cells or endothelial progenitors or endothelial cells.Cells that are identified in the CD45/CD19/CD14 panel would be definedas CD45+/CD19+=B cells, CD45+/CD14+=monocytes, and cells that areidentified in a CD45/CD271 panel would be defined as CD45−/CD271+ areMSCs.

In specific embodiments, a final dosage of at least 100 million BMC istransplanted to the patient.

In a third aspect of the present invention, an antibody panel comprisesa first antibody or other substance which specifically binds to a firstbone marrow cell (BMC) marker and a second antibody or other substancewhich specifically binds to a second bone marrow cell (BMC) marker. Thefirst and second markers are characteristic of the suitability of theBMC for transplantation to a heterologous tissue site in the patient.

The at least two antibodies are specific for CD markers selected fromthe group consisting of CD14, CD19, CD34, CD45, CD133, CD271, with twoindividual antibodies typically being specific for at least CD34 andCD133, respectively, and often with three individual antibodies beingspecific for at least CD19, CD34, and CD 133, respectively. Theantibodies in the panel may be suitable for use in flow cytometry or forany other conventional cell surface marker screening methodology.

As used herein, the phrase “bone marrow cells” (BMC) refers to the bonemarrow mononuclear cell fraction.

As used herein, the phrase “autologous BMC” refers to BMC harvested fromthe patient to be treated with the minimally processed BMC.

As used herein, the phrase “minimally processed BMC” refers to freshaspirated bone marrow concentrated using gravity centrifugation at thebedside. Here bone marrow mononuclear cells are concentrated by densityusing either a system such as the Biosafe SEPAX system, the BiometMarrowstim System, Thermogenesis ResQ System, and the Terumo Harvestsystem, or the Syngen's SyngenX 2000.

As used herein, the phrase “phenotype profile,” refers to geneexpression profiling where RNA or protein in a sample is measured, DNAsequencing, surface marker assessment of cells such as the cluster ofdifferentiation immunophenotyping performed on mononuclear cells,ability of the cells to meet a functional assay such as the ability todivide or proliferate, the ability to express antigens or the like invitro, or combinations thereof.

As used herein, “CD profile” refers to the number or concentration ofcells with different CD characteristics in the patient BMC. CD markersof interest include but are not limited to CD14, CD19, CD45, CD34,CD133, and CD271. In some embodiments, any one or more of theapproximately 400 known CD markers may find use.

As used herein, the term “transplantation” includes all forms of tissueheterologous implantation of the BMC cells to other tissue sites. Formsof transplantation include, injection, infusion, surgical implantation,and the like. Other heterologous tissue sites include cardiac myocardiumand other tissue and other tissue, such as the muscles in the legs ofpatients with limb ischemia.

As used herein, the term “heterologous” refers to a transplantation sitein the patient other than the bone marrow that is the source of the BMCor implantation into the marrow of another bone within the body.Examples of heterologous tissue sites have been provided above.

As used herein, the term “antibodies” will refer to conventionalpolyclonal and monoclonal antibodies as well as recombinant antibodies,antibody fragments from any of these sources, and other specific bindingsubstances which can specifically bind to cell surface marked with aspecificity and affinity equivalent to natural antibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph comparing intracoronary artery delivery, straightneedle intramyocardial delivery, and helical transendocardialintramyocardial delivery of ¹⁸F-FDG labeled bone marrow derivedmononuclear cells in a swine heart.

FIG. 2 shows calculations for estimating total doses delivered and theeffective dosages of CD34⁺, CD133⁺ and CD19⁺ cells.

FIG. 3 shows the calculated estimated effective doses retained one hourafter delivery for CD34⁺, CD133⁺ and CD19⁺ cells in the four clinicaltrials shown in FIG. 2.

FIGS. 4 and 5 show the minimum number of cells needed per ml of bonemarrow aspirate harvested from the patient to achieve an effectivedosage.

FIG. 6 shows the minimum number of cells needed per ml of bone marrowaspirate harvested from the patient to achieve an effective dosage withtrans-endocardial delivery.

FIG. 7 shows a partial response of a patient population.

FIG. 8 shows the results required for a diagnostic assay output toexceed an effective therapeutic dosage form.

FIGS. 9 and 10 show a plots of the change in left ventricular ejectionas a function of the CD133⁺ and CD34⁺ cell counts, respectively.

DETAILED DESCRIPTION OF THE INVENTION

Delivery routes and the retention of ¹⁸F-FDG labeled bone marrow derivedmononuclear cells in the swine heart after 1 hour, as measured by PET-CTare summarized in FIG. 1. Intracoronary artery delivery, straight needleintramyocardial delivery, and transendocardial intramyocardial deliveryof ¹⁸F-FDG labeled bone marrow derived mononuclear cells in the swineheart were compared. Here, one hour after delivery of autologous bonemarrow mononuclear cells at a concentration of 100 million cells per cc,retention rates of 1% for intra coronary artery delivery, 6% forstraight needle transepicardial delivery, and 18% for transendocardialdelivery using the Helical Infusion catheter system were observed. Forlocal delivery of cells, it is the local time course of regionaldistribution that defines the dosage and not the amount delivered. Thisis different for systemic delivery where dosage is often defined as thebioavailability of a therapeutic agent in the circulating blood. As manybiologic agents are infused into the blood vessels, systemic dosageconcentration is the same as the delivered dosage divided by the bloodvolume. Thus for local delivery, where agents egress from the tissueafter delivery, one measure of effective dosage is the amount of agentdelivered that stays at a local delivery site at a period of time afterdelivery. By this measure, the same number of mononuclear cellsdelivered transendocardially with the helix transendocardial deliverysystems, straight needle intramyocardial delivery system, andintracoronary artery delivery route will result in different effectivedosages due to the different retention profiles for these deliveryparadigms. The phrase “effective dosage” for cell therapy is defined asthe minimum dosage necessary for the cells to be retained in the tissueone hour after delivery,

Patient specific cellular source material is also an issue. Patientsfrom the FOCUS-HF Trial (Am Heart J 2011; 161:1078-1087.e3.) received 30million autologous bone marrow cells with an intramyocardial straightneedle approach, but likely had an effective dosage of 1.8 Million cells(30 million×6%) using the retention rate discussed above. The resultsshowed that “In fresh bone marrow samples, hematopoietic and mesenchymalclonogenic assays showed decreased progenitor cell activity in ourpatient population.” and “Autologous BMMNCs obtained from patients withstable ischemic HF have a lower hematopoietic proliferative capacity(CFU-GM) and lower migratory capacity (in response to VEGF andstromal-derived factor 1) when compared with the proliferative andmigratory capacities of healthy controls.” These patients did not havesignificant improvement in myocardial function when the overall patientpopulation was considered. However, in a post hoc analysis, youngerpatients (≤60 years) who are believed to have greater potency in theircells, were observed to have significantly improved MVO₂.

Patients from the FOCUS-CCTRN Trial (JAMA 2012; 307(16):doi:10.1001/jama.2012.418) delivering 100 million autologous bone marrowcells using the same delivery methodology as the FOCUS-HF Trial,received an effective dosage of 6 million bone marrow mononuclear cells(100 million×6%) per the retention rate discussed above. Similarly, theFOCUS-CCTRN Trial patients did not show improvements in the primaryendpoints. However, a regression analysis showed that higher CD34 cellor CD133 cell counts were associated with greater absolute unit increasein LVEF. Here, the range of CD34 was 0.5% to 6.9% (SD, 1.2%) with anaverage value of 2.6%. Additional analysis was claimed to suggest thatevery 3% higher level of CD34 cells was associated with on average a3.0% greater absolute unit increase in LVEF in a multiple variable modelthat included age and treatment as predictor variables. An analogouscomputation for CD133 cells (range, 0.1%-3.6%; SD=0.62 with an averagevalue of 1.2%) revealed that every 3% higher level of CD133 cells wasassociated with on average a 5.9% greater absolute unit increase inLVEF.

Losordo 2011, showed efficacy in a setting of refractory angina withautologous and significantly processed dosage of 1×10^5 CD34+ cells perKG (a calculated dose of 7,000,000 cells with an assumed 70 Kg averageweight patient) for the clinical indication of chronic myocardialischemia with a positive improvement in angina frequency and exercisetolerance time. This study used a straight needle intramyocardialdelivery paradigm, suggesting that approximately 6% of the dosage wasretained (CD34-R) in the target tissue one hour after delivery. Assuminga calculated dosage of 7,000,000 CD34 cells delivered in a 70 KGpatient, the resulting effective dosage would be 420,000 CD34+ cells ifthe retention rate of 6% is used.

De la Fuente et al. (Transendocardial Autologous Bone Marrow inMyocardial Infarction Induced Heart Failure, Two Year Follow up in anOpen Label Phase I Study, Eurointervention 2011; 7:805-812.) delivered100 million cells using a helical needle intramyocardial approach,resulting in an effective dosage of 18 million cells at the local targetsites in the peri-infarcted region of the heart. Assuming that averagepercent of 2.6% CD34 cells present in bone marrow mononuclear cellsreported in FOCUS-CCTRN, the calculated number of CD34 cells in 18million bone marrow mononuclear cells retained at the local target sitesis 468,000 cells, which is roughly the same number of CD34+ cells as theLosordo 2011 study, shown to be therapeutically efficacious. Both theFOCUS-HF and the FOCUS-CCTRN trials had significantly lower calculatedeffective doses of 1.8 million and 6 million autologous bone marrowderived mononuclear cells at the local target sites of delivery.

FIG. 2 shows the calculations for the estimation of the total dosesdelivered and the effective dosages of CD34, CD133 and CD19 cell countsretained at one hour after delivery (CD34-R, CD133-R, CD19-R) for fourclinical trials (ACT34-CMI, TABMMI, FOCUS-HF and FOCUS-CCTRN) using thepercent cell retention results based on the delivery routes used in therespective clinical trial as shown in FIG. 1. Here, the percentage ofcells having CD34, CD133, and CD19 surface markers were all analyzed.The percent numbers in columns 3, 4 and 5 were either reported averagenumbers from published results for the trials or estimates taken fromthe closest available data. Estimates are detailed in the table withan * or a ^(¥). For the ACT34-CMI 2011 study, the estimate of 100% CD34+cells was used because the CD34+ cells were first isolated frommobilized autologous peripheral blood cells and then expanded to thespecific total cell dosage needed for injection. In this case, no datawas shared on the purity of the formulation in question and therefore,it was assumed to be 100% purely CD34+ cells. For the TABMMI 2011 study,the percentage of CD19 cells was assumed to be the same as that in thesimilar patient population treated in FOCUS-HF 2011, the only clinicaltrial that reported on the percent of CD19 cells in the autologous bonemarrow mononuclear cell fraction used in the injection procedure.Likewise for FOCUS-HF 2011, the percentage of CD133 cells was assumed tobe similar to that in TABMMI 2011 as we wanted to be more conservativein our estimates. However, one could also assume that the same 1.21percent of CD133 could be present in FOCUS-HF 2011 as was measured inFOCUS-CCTRN 2012 for a higher threshold of CD133. This would beequivalent to 363,000 CD133 cells in the autologous bone marrowmononuclear cells fraction injected in the FOCUS-HF study with anestimated 21,780 CD133 cells retained based on the straight needleintramyocardial route of delivery. FIG. 3 shows the calculated estimatedeffective doses retained one hour after delivery for CD34, CD133 andCD19 cells in the four clinical trials as detailed in FIG. 2.

From these results, we could consider four different scenarios asdetailed in Examples 1 to 4. A simple signature that can be specifiedtoday is that any patient who has sufficient CD133 cells, and/or CD34cells, and/or CD19 cells to achieve an efficacious therapeutic dosageform are likely to yield a far more positive response to therapy basedon the best clinical data available today (Perin 2012) and anycombination of one or more of these cell count thresholds can be setdepending on the patient population and their level of therapeuticresponsiveness.

Examples 1 to 3 below detail the development of potency assays forautologous bone marrow therapy to treat cardiac disease based on abiomarker panel which characterizes that a patient's cells will meet athreshold dose for multiple cell types that enhance potency. Effectivedosage in the target delivery sites is dependent on delivery efficiencyand whether the patient would have enough of these cells available intheir bone marrow harvest to generate the final intended effectivedosage.

EXAMPLE 1

This example describes the selection of patients for autologous tissuetherapies based on the analysis of small tissue samples from thepatients in advance of the therapy. Such tissue samples and patientselection is determined based on pre-defined thresholds of cellularcharacteristics such as number of cells per unit volume of tissue,functional capacities, gene expression profiling, and/or cell surfacemarkers such as the levels of known cluster of differentiation (CD)surface markers of the constituent cells. The use of CD markers forimmunophenotyping is well known in the art and described in commonlyavailable resources such as:http://en.wikipedia.org/wiki/Cluster_of_differentiation. CD thresholdsselected are dependent on the disease being treated and are determinedbased on past studies where patient improvement has been correlated withthe levels of certain CD cell surface markers or other biomarkers intheir tissue. In the case of heart failure patients of ischemicetiology, CD 19, CD34, and CD 133 markers are commonly looked at surfacemarkers. In this example, potential successful patients with a higherprobability of being a positive responder, can be identified asappropriate for treatment by selecting candidates with sufficient CD133,CD34, and CD19 cell counts to achieve an efficacious therapeutic dosagevia their preferred delivery route. The effective dosage was calculatedas the retained cells one hour after delivery in FIG. 2 and wasdetermined to be at least 420,000 CD34+ cells (highest in Losordo 2011),72,000 CD133 cells (highest in FOCUS-CCTRN 2012), and 144,000 CD19 cells(highest in FOCUS-CCTRN 2012; it should be noted that the percent ofCD19 in the autologous bone marrow mononuclear cells used to estimatethe CD19 cell count in FOCUS-CCTRN was extrapolated from FOCUS-HF)retained one hour after delivery. The assumptions used in this Example 1lie in using the estimates of CD34 cells from the ACT34-CMI trial and inno cell losses due to cell processing. Further, assuming that a full 60ml of bone marrow aspirate is processed from the patient, to achieve theeffective dosage of at least 420,000 CD34 cells, and/or 72,000 CD133cells and/or 144,000 CD19 cells using the Helix transendocardialdelivery system, the minimum number of cells needed to be present per mlof bone marrow aspirate harvested from the patient to achieve such aneffective dosage would be 38,889 CD34 cells, 6,722 CD133 cells and13,333 CD19 cells (FIG. 4). Therefore, one could specify that a simplesignature of a patient would be for him or her to have at least 38,500CD34 cells, and/or 6,500 CD133 cells, and/or 13,000 CD19 cells per ml ofbone marrow harvest. Patients who had this threshold signature in asimple small bone marrow aspirate days in advance of a procedure, areexpected to have greater responsiveness to therapy. In this preferredembodiment, the patient would have 5 cc of marrow aspirated on day 1which would have the thresholds measured using flow cytometry andreported back to the physician on day 3. Once the physician has thisinformation, the patient could be scheduled for therapy which involvesharvesting 60 ml of bone marrow from the iliac crest, concentrating itat point of care to a 5 cc volume using gravity centrifugation fordelivering therapy, and delivering therapy at ten sites in 0.5 ccaliquots via transendocardial intramyocardial delivery using a catheterwith a helical needle at its distal end and a means to confirmengagement with the heart using fluoroscopy. This preferred embodimentis in itself merely a broad example as other delivery systems with otherefficiencies could be used to calculate different dosages. Furtherpatients who do not meet threshold could receive a modified higherdosage so that they meet threshold.

EXAMPLE 2

Here we calculate the thresholds for a cardiac therapy as in Example 1.In Example 2, the assumptions are changed to use the effective dosage ofat least 162,600 CD34 cells from the FOCUS-CCTRN clinical trial whichuses autologous bone marrow mononuclear cells isolated using densitygradient centrifugation with Ficoll Paque, instead of the estimated CD34cells from the ACT34-CMI trial. The same assumption of no cell lossesdue to cell processing was applied. For our purpose, any reagent such aslymphocyte separating medium having the same characteristics and densityas Ficoll Paque could also be used in the cell processing. Therefore,assuming that a full 60 ml of bone marrow aspirate is processed from thepatient, to achieve the effective dosage of at least 162,600 CD34 cells,and/or 72,000 CD133 cells and/or 144,000 CD19 cells using the Helixtransendocardial delivery system, the minimum number of cells needed tobe present per ml of bone marrow aspirate harvested from the patient toachieve such an effective dosage would be 15,056 CD34 cells, 6,722 CD133cells and 13,333 CD19 cells (FIG. 5). In this case, one could specifythat a patient would qualify by having a simple signature of at least15,000 CD34 cells, and/or 6,500 CD133 cells, and/or 13,000 CD19 cellsper ml of bone marrow harvest.

EXAMPLE 3

In Example 3, instead of isolating autologous bone marrow mononuclearcells using density gradient centrifugation with Ficoll Paque or othersimilar reagent, we use a point of care cell processing system whichconcentrates autologous bone marrow or blood-derived mononuclear cellsusing the same technique of density gradient centrifugation without theaddition of Ficoll Paque. In this case, we further assume 20 to 50percent cell losses due to the cell processing. Therefore, assuming that54 ml of bone marrow aspirate is processed from the patient (it shouldbe noted that 54 ml of bone marrow aspirate is used here to take intoaccount the 6 ml of heparin or other anticoagulating agent used totypically make up the full volume of 60 ml during bone marrow harvest),to achieve the same effective dosage of at least 162,600 CD34 cells fromthe FOCUS-CCTRN clinical trial, and/or 72,600 CD133 cells, and/or144,000 CD19 cells using the Helix transendocardial delivery system, theminimum number of cells needed to be present per ml of bone marrowaspirate harvested from the patient to achieve such an effective dosagewould be 20,910 to 33,457 CD34 cells, and/or 9,336 to 14,938 CD133cells, and/or 18,519 to 29,630 CD19 cells (FIG. 6). In this case, if wewant to be conservative, we could specify that a patient would need tohave a simple signature of at least 33,500 CD34 cells, and/or 15,000CD133 cells, and/or 30,000 CD19 cells per ml of bone marrow harvest. Onthe other hand, we could assume that the point of care cell processingsystem is a lot more efficient and that 80% of the cells processed aretypically recovered from the system such that a patient would need tohave at least 21,000 CD34 cells, and/or 9,500 CD133 cells, and/or 19,000CD19 cells per ml of bone marrow harvest. Any combination of number ofCD34 cells, CD133 cells and CD 19 cells within the 50 to 80% recoveryrange could potentially be used to pre-specify thresholds for a patientbased on the known efficiency of the system used. This efficiency ofcell recovery rate could already be known or could be determined orchanged prospectively after the processing of a number of samples.

EXAMPLE 4

An embodiment of this invention whereby we consider that the patientwould potentially be responsive to a therapy that may or may not beautologous merely based on the levels of certain CD markers in theirbone marrow aspirate. Bone marrow cells respond to therapies thatstimulate the expression of chemokines in tissues. For cardiacapplications, allogeneic mesenchymal stem cells, allogenic mesenchymalprecursor cells, and allogeneic cardiosphere derived cells are all inclinical development and reportedly act by a mechanism of action inwhich paracrine signaling triggers a response in the patient. Theseresponses very likely involve the cells of the immune system anddeveloping a responsiveness assay has significant value for thesepatients. This is more in line with the classic strategy ofpharmacogenomics, but here we are looking specifically at theresponsiveness of the gene expression, surface markers, or cell countsof cells from the bone marrow. This strategy may be used similarly withautologous therapies that are either highly processed or minimallyprocessed. Looking at the FOCUS-CCTRN trial only as an example of apatient population that almost responded, the potential responders wouldhave to have at least 27,100 CD34 cells, and/or 12,100 CD133 cells,and/or 24,000 CD19 cells per cc of bone marrow aspirate (FIG. 7). Thisis the half of the population with the most potent cells.

EXAMPLE 5

A therapy of 200 Million Autologous Bone Marrow Mononuclear CellsConcentrated using Gravity Centrifugation with the Cells from 60 cc ofbone marrow aspirate is to be developed. The results required for thediagnostic assay output to exceed an effective therapeutic dosage formwith at least 420,000 CD34 cells, 144,000 CD19 cells, and 72,600 CD133cells remaining in place sixty minutes after delivery are shown in FIG.8 for both a 1 cc and a 10 cc aspirate. If the efficiency of deliverywas 100%, most patients would likely qualify. The number of patientsthat qualify decrease significantly as the efficiency of deliverydecreases. This erodes the value of the therapy. FIG. 8 shows the cellcounts that must be present to achieve the therapeutic dosage with theperfect 100% retention in the tissue one hour after delivery, as well aswith the percent retention achieved with three leading delivery systems:transendocardial delivery with the helical infusion system (BioCardia,Inc. San Carlos, Calif.), a straight needle system such astransepicardial syringe delivery system, and an intracoronary arteryinfusion system using a standard balloon catheter. Other deliverysystems and methods used to achieve different retention results one hourafter delivery can be within the scope of this invention. For example ifextreme care is used and a transepicardial needle is inserted at asignificant angle, cell retention in the tissue can be greater. Manyother variables which affect retention including timing of delivery withrespect to the heart cycle, volume of delivery, target tissue fordelivery etc., can be modified.

A preferred embodiment here is the use of a diagnostic assay to selectpatients who have at least 21,000 CD34⁺ cells, 9,500 CD133 ⁺cells, and19,000 CD19⁺ cells per cc bone marrow aspirate from their iliac crest toreceive the delivery of one to ten cc of cells concentrated from afifty-four (54) cc. harvest of bone marrow aspirate by a point of carecell concentration system The cells are then delivered to up to fifteenlocations adjacent to damaged tissue using the Helix Infusion System. Itis noted that immature CD19 cells also express CD34⁺ cells (Loken M R etal, Flow Cytomeric Analysis of Human Bone Marrow II. Normal B LymphocyteDevelopment, Blood Vol 70, No 5; 1987 pp1316-1324.). Patients who failto meet this threshold may not be selected for treatment.

Clearly, these threshold values can be calculated based on theefficiency of delivery of any delivery platform. For less efficientdelivery systems than the Helix transendocardial delivery system, suchas the straight needle endocardial or straight needle epicardialsystems, an operator could pull more marrow for processing to meet therelative threshold for effective dosages of these cell types. However,as the volume extracted increases, the pain and discomfort to thepatient increases. Further, thresholds of one or more cell surfacemarkers may be selected for this and other types of therapies.

The processing platform efficiency must also be considered. With theHelix Transendocardial Delivery System and the Marrow Stim processingplatform for processing 60 cc of marrow, a final volume of 6 ccconcentrate can be obtained. If only 5 cc of marrow at a concentrationof 40 million cells per cc is to be used for therapeutic purposes, theaspirate thresholds would be higher as a portion of the cells (1 cc)will not be delivered. The thresholds are easily calculated as has beendescribed.

As one treats and follows many patients tracking details on the cellcharacteristics will help to enhance understanding of threshold levelsand new thresholds may be set. Such in vitro multivariate analyte assaycan be run as a homebrew assay in a Clinical Laboratory Institute ofAmerica without FDA clearance or can be submitted for a deNovo 510(k)pathway for FDA approval.

Other trial result thresholds may be set. FIG. 9 shows a plot of thechange in left ventricular ejection as a function of the CD133 cellcounts delivered from action from the TABMMI trial. There is a cleartrend in the patients with higher CD133 cells in that they have moreimprovement and this is in agreement with the post hoc analysis reportedin the literature in FOCUS-CCTRN 2012. Here the threshold of 30,000cells delivered with Helix is set in which all patients above thisthreshold are responders. This is shown by line 501. This is thethreshold set based on delivery with the Helical Infusion Catheter andwith a 18% retention corresponds to a 7500 CD133 cell effective dosagein autologous concentrated bone marrow mononuclear cells. This can beused to define the thresholds that must be present in aliquots takenfrom the patient before therapy as shown in FIG. 8.

It should be clear that additional data which already exists but whichhas not been published could be used to define a new threshold, and thata threshold could be set where 100%, 90%, 80%, or even only 70% of thepatients respond based on other parameters. Further, merely runninglarger trials and tracking cell phenotype and response to therapy willenable these thresholds to be revised and validated in subsequentprospective clinical trials if needed. Further, as noted, increasing thedosage or modifying delivery to enhance retention and therapeutic dosagecan enable modifications in a therapeutic strategy to enable morepatients to have potential to receive therapeutic benefit. In this way,one may tailor the therapy to a specific group of patients or even aparticular patient in a personalized medicine approach so that thepatients would receive optimal therapeutic benefit.

Based on the patients treated in FIG. 4, at 100M cells with helix thisthreshold criteria excludes more patients from therapy than a 200M cellswith helix using this same threshold criteria.

As similarly shown in FIG. 10, using the dosage of Losordo to set athreshold of 420,000 for effective dosage of CD34⁺ cells with Helix inTABMMI 2011 would correspond to 420,000 divided by the efficiency ofdelivery of 0.18 results in the need to deliver 2,333,333 CD34+ cells.This would exclude 13 of 17 patients at dosage of 100M cells with helixas assessed from the results with the TABMMI trial shown as line 701.However at a dosage of 200M cells delivered using the same patients withhelix the threshold line would move to the left 702 and all but threepatients would meet the threshold. Here we assume that there are nosynergistic or antagonistic effects of other cells in the autologousbone marrow concentrate, which will either be validated or challenged bydata obtained in clinical trials. Here a simple 50 ml aspirate and pointof care processing at a cost of a few hundred dollars has potential toreplace a CD34⁺ therapy obtained by GCSF stimulated release of the cellsfrom the bone marrow, apheresis of the patient's blood to extract thecells, and isolation of only the cells of interest at a cost of $10,000to $25,000. If there are synergistic effects of the other cells it mayalso prove to be a better therapy. If antagonistic, additional data maybe used to predetermine thresholds and dosages to deliver an effectivedosage of autologous minimally processed cells.

Of note, although the patients in TABMMI 2011, FOCUS 2011, and FOCUS2012 were of similar phenotypes, the first had no active ischemia andthe latter two had mostly active ischemia (FOCUS 2012 was changed toallow non-ischemic patients into the trial midway through). The CD 34⁺cells available in the bone marrow of these different patientpopulations varied greatly. CD34⁺ cell counts per cc of bone marrowaspirate in FOCUS-CCTRN 2012 exceeded that in TABMMI 2011 and FOCUS-HF2011. Clearly, precision in establishing measurement techniques forsetting these thresholds will be important in larger trials and futuredevelopment. Understanding that cells are counted the same way in aseries of trials and knowing both average and standard deviations forthreshold setting will be important.

CD19 expressing cells have also been detailed as important potentialtherapeutic agents. Here, therapeutic threshold is set at the reportedmedian average for these cells in the tissue of patients with heartfailure as reported in FOCUS-HF 2011 (a negative trial) with the threefold increase in dosage and threefold increase in efficiency of deliveryas reported by the inventors in TABMMI 2011. This effective dosage valuewould be 600,000 cells. The selection of this threshold is expected toeliminate half of the patients at 100M cells used in TABMMI, but a muchsmaller number of patients at a dosage of 200M cells.

A patient can be selected for a predefined dosage form by assessing thesuitability of their autologous tissues to meet predefined effectivedosages that can be achieved using available delivery systems.Suitability can be assessed using cell surface markers as detailed here,using gene expression analysis as detailed in 2010/0127342, combiningthese techniques, incorporating other molecular measurement techniques,such as the ease with which they express a particular surface antigen,and even incorporating details of the phenotypic characteristics of apatient and their disease state. For example, the thresholds for one ormore CD cell surface markers in combination with one or more geneexpression values for one or more patient phenotypic characteristics islikely to evolve over time. Such clinical phenotypic criteria thatincludes race, sex, age, diet, and other risk factors may beincorporated in such an analysis as independent variables. The eleganceof this method of developing thresholds of efficacy for autologous cellbased therapies is that autologous therapies can evolve over time as welearn more of the potential of these cells to treat known and unknowndiseases.

A therapeutic dosage form may also be set by one or multiple of these CDcell surface markers, other cell surface antigens, proteins and/orgenes, as well as functional assays. Selecting patients for therapy andsetting their required dosages to be delivered via specific deliverysystems and delivery routes based on what appears to be retained foreffective local dosage one hour after delivery depending on theiravailable autologous tissue and available delivery technology is a noveltherapeutic approach and will result in far less expensive and moreeffective products for the treatment of patients.

The methods for analyzing multiple variables include principalcomponents analysis, linear discriminant analysis (LDA, StatSoft, Inc.),logistic regression (SAS Institute, Inc.), prediction analysis ofmicroarrays (PAM) voting, classification and regression trees (TreeNet,Salford Systems), Random Forests, nearest shrunken centroids andk-nearest neighbors.

Once a small set of variables (cell surface markers, gene expressionthresholds, protein expression levels, and/or phenotyping patientinformation) are selected, confirming their potential to act as asignature for therapeutic efficacy may require tuning and will requireclinical validation. Tuning involves defining an equation with thevariables to optimally separate the responders from the non-respondersand testing involved validation of the test results in a clinical study.Some groups (Deng et al American Journal of Transplantation 2006; 6:150-160) have used these techniques to develop a linear discriminantanalysis system which is relatively simple, other approaches are alsopossible which do not reduce the output to a simple scalar score.

One approach involves ranking the variables for their ability toindependently separate the groups (responders versus non-responders) andconstructing a tree shaped map that ranks the variables based on theirability to separate the responders from the non-responders. Eachvariable creates three scenarios: (A) a probability greater than 50+X%that patient is a responder, (B) a probability greater than 50+X% thatpatient is a non-responder, or AB indeterminate in which neither A nor Bis true. X may be selected and need not be that large for this techniqueto work well. The first variable that separates with the greatest degreeof confidence for a given X then defines three classes: A1, B1, and AB1.This is the trunk of the graphical tree. The second variables build onthis scoring and one gets nine separate classifications possible: A1A2,A1B2, A1AB2, B1A2, B1B2, B1AB2, AB1A2, AB1B2, AB1AB2. This is the firstset of branches off of the trunk branches. A third variable similarlyresults in 81 independent states that may be assessed and so on. This isthe third set of branches. Such a graphical readout is possible for tensof variables which with color coding of the As and Bs one can begin toreadily interpret patterns where there is high confidence on therapeuticeffectiveness without discarding important data that may be useful forfuture analysis and interpreted graphically for up to 20 rankedvariables (including quality control genes such as plant genes, orsurface markers that appear on all cells). If X is selected to bevanishingly small, a binary score is possible that makes tracking thedecision aspects of each variable contributor to the algorithm muchsimpler. As yet undefined algorithms may be employed in this inventionas there is significant ongoing work in this area.

For bone marrow cells to treat ischemic heart failure, the thresholdsdisclosed here can be validated by use of these thresholds as inclusioncriteria for clinical trial design. A clinical trial that achievestherapeutic efficacy based on the use of these criteria, would beconsidered validation of the criteria. Over time the criteria may bemodified in further investigational clinical studies. For example, if asuccessful validation suggests that a large portion of patients arebeing excluded from therapy because they typically don't have onevariable's threshold met, a clinical non-inferiority study could be runto assess the ability to lower the threshold of one or more variables.Similarly, if there is a new variable that is believed to enhancetherapeutic potential, trials could be performed to assess patientbenefit based on a modified threshold including that new variable,without invalidating the previous thresholds set. This includes all thecharacterized CD molecules generated by studies of leukocyte surfacemolecules, organized through a series of international workshops knownas the Human Leucocyte Differentiation Antigens (HLDA) Workshops. A fulllist of current characterized CD molecules is available on the HumanCell Differentiation Molecules (HCDM) website (http://www.hcdm.org), anorganization that runs the HLDA Workshops and names and characterizes CDmolecules (http://www.uniprot.org/docs/cdlist). These CD molecules are amajor focus and are widely used in research where their up regulation ordown regulation have been found to play a significant role indifferential diagnosis, monitoring and treatment of many diseases. Atpresent, CD markers CD1 to CD364 are current.

The detailed description of thresholds for this invention focus onischemic heart failure patients and autologous bone marrow mononuclearcells. However, other autologous cell types for a variety of indicationscould benefit from this therapeutic strategy—both for initial productapprovals and improvement in efficacy over time. This includesadipose-derived regenerative cells (Cytori), expanded CD34+ cells (CellProThera), cord blood cells (Perkin Elmer), expanded autologousmulticellular therapy including primarily autologous mesenchymal stemcells (Aastrom Biosciences Inc.), expanded autologous bonemarrow-derived mesenchymal cardiopoietic cells (Cardio3 Biosciences),and CD34+, CD133+, and CD19+ enriched bone marrow or blood-derivedprogenitor cells (Baxter, Miltenyi, and AC Therapeutics) for theindications of cardiovascular disease (ischemic heart failure,non-ischemic heart failure, diastolic heart failure, systolic heartfailure, chronic myocardial ischemia and ischemic heart failure withoutactive ischemia or chronic myocardial infarction), autoimmune diseasessuch as Lupus, inflammatory bowel diseases such as Crohn's andulcerative colitis, diabetes, and renal diseases, as well asimmunological strategy for treating cancer with harvested andre-administered autologous immune cells. For cord blood cells, taken atbirth, the diagnostic element towards efficacy would be performed oncells from the banked cell samples intended for therapy. This could bedone with other cell types as well.

All patents, publications, and abstracts cited above are incorporatedherein by reference in their entirety. It should be understood that theforegoing relates only to preferred embodiments of the present inventionand that numerous modifications or alterations may be made thereinwithout departing from the spirit and the scope of the present inventionas defined in the following claims.

While the above is a complete description of the preferred embodimentsof the invention, various alternatives, modifications, and equivalentsmay be used. Therefore, the above description should not be taken aslimiting the scope of the invention, which is defined by the appendedclaims.

What is claimed is:
 1. A method for screening a patient with chronicmyocardial ischemia or heart failure of ischemic etiology to determine alikelihood that the patient's autologous bone marrow cells will besuitable for transplantation to a heterologous tissue site to treat thepatient's chronic myocardial ischemia or heart failure of ischemicetiology, said method comprising: providing a sample of bone marrowcells (BMC) from the patient with chronic myocardial ischemia or heartfailure of ischemic etiology; obtaining, with a cell counter, aphenotypic profile of the BMC in the sample, wherein the phenotypicprofile comprises a CD profile comprising the concentrations of CD19⁺,CD34⁺, and CD133⁺ cells; and determining, with the cell counter, whetherindividual concentrations of CD19+, CD34+, and CD133+ cells in the CDprofile each meet or exceed threshold concentrations of CD19⁺, CD34⁺,and CD133⁺ cells, respectively, to identify the BMC from the patientwith chronic myocardial ischemia or heart failure of ischemic etiologyas being suitable for transplantation to the heterologous tissue sitewithout enriching particular phenotypic population(s) of the BMC totreat the patient's chronic myocardial ischemia or heart failure ofischemic etiology, wherein the BMC from the patient with chronicmyocardial ischemia or heart failure of ischemic etiology are identifiedas suitable for said transplantation if each of the determinedindividual concentrations of CD19+, CD34+, and CD133+ cells meet orexceed the threshold concentrations of CD19+, CD34+, and CD133+ cells,respectively.
 2. A method as in claim 1, wherein the CD profile consistssolely of CD19⁺, CD34⁺, and CD133⁺ cells.
 3. A method as in claim 1,wherein the concentrations comprise threshold concentrations of 21,000CD34⁺ cells, 9,500 CD133 ⁺cells, and 19,000 CD19⁺ cells.
 4. A method asin claim 3, wherein the concentrations comprise thresholdconcentrations, at least 27,100 CD34⁺ cells/ml, 12,100 CD133⁺ cells/ml,and 24,000 CD19⁺ cells/ml.
 5. A method as in claim 1, wherein the sampleof bone marrow cells (BMC) comprises bone marrow aspirate.
 6. A methodas in claim 5, wherein the bone marrow aspirate is treated only with ananticoagulant.
 7. A method as in claim 1, wherein the cell countercomprises a flow cytometer.